Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio)

A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α₁-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of α₁-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of α₁-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum α₁-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically. Received June 4, 1998; accepted September 10, 1998.     

鲤鱼微卫星分子标记的筛选

微卫星 (ｍｉｃｒｏｓａｔｅｌｌｉｔｅ)是近十几年来发展起来的一种新的分子标记 ,它是指以少数几个核苷酸 ( 1～ 6个 )为单位多次重复的简单序列 ,以双核苷酸重复最为常见 ,而其中又以 (ＣＡ/ＧＴ) ｎ 居多。由于微卫星在真核生物基因组中是随机分布的 ,而作为分子遗传标记又有着非常高的多态性和共显性 ,因此在构建遗传连锁图谱时备受青睐 (Ｂｒｏｏｋｅｔａｌ ,1994 )。目前 ,在人类和多种动物中已经构建了以微卫星为主的遗传连锁图谱。但在鲤鱼等水产动物的遗传连锁图谱中 ,微卫星分子标记还较少 (孙效文和梁利群 ,2 0 0 0 )。为了摸索…     

Carp (Cyprinus carpio) as a powerful invader in Australian waterways

1. The invasion of carp (Cyprinus carpio L.) in Australia illustrates how quickly an introduced fish species can spread and dominate fish communities. This species has become the most abundant large freshwater fish in south‐east Australia, now distributed over more than 1 million km². 2. Carp exhibit most of the traits predicted for a successful invasive fish species. In addition, degradation of aquatic environments in south‐east Australia has given them a relative advantage over native species. 3. Derivation of relative measures of 13 species‐specific attributes allowed a quantitative comparison between carp and abundant native fish species across five major Australian drainage divisions. In four of six geographical regions analysed, carp differed clearly from native species in their behaviour, resource use and population dynamics. 4. Climate matching was used to predict future range expansion of carp in Australia. All Australian surface waters appear to be climatically suitable for carp. 5. This assessment strongly reinforces the need for immediate management of carp in Australia to include targeted control of human‐assisted dispersal, such as use of carp as bait by anglers, distribution to new locations by anglers and the use of the ‘Koi’ strain in the aquarium industry. 6. Given their historical spread, dispersal mechanisms and ecological requirements, the expansion of carp across most of the remainder of Australia is to be expected.     

转反义sGnRH基因败育鲤鱼的性成熟诱导

采用反义转基因技术已成功获得整合CAsGnRHpc-antisense基因的转基因鲤鱼,简称AS-sGnRH F0鲤鱼.通过挤压1年龄AS-sGnRH F0鲤鱼的腹部,发现该群体中有精液/无精液个体比例为33/69,与对照组鲤鱼中有精液/无精液个体比例接近1：1相比存在显著差异.通过注射鲤鱼垂体抽提物诱导无精液AS-sGnRH F0鲤鱼恢复繁殖功能,获得3尾育性被恢复的AS-sGnRH F0鲤鱼.进一步的解剖观察发现,在无精液AS-sGnRH F0鲤鱼群体中,完全无性腺的AS-sGnRH F0鲤鱼比例约31.3%.根据直观的挤精液和解剖学观察,推测在AS-sGnRH F0鲤鱼群体中,雄鱼：雌鱼：＂脂肪型＂性腺个体的比例大约为35：45：20%.受精卵发育12 h后,统计A、B、C系AS-sGnRH F0鲤鱼精子的受精率分别为73.94、59.45和64.05%.从A、B、C系杂交后代F1分别随机取样100尾,PCR检测的阳性率分别为0、78.1和87.8%.研究结果表明通过补充外源激素可以使促性腺激素分泌不足而不育的AS-sGnRH F0鲤鱼恢复具有产生成熟配子的能力.     

烟酸诺氟沙星在松浦镜鲤体内的药动学特征

结合单纯聚集法和二步法,应用高效液相色谱(HPLC)技术研究了分别以10、30、60 mg/kg剂量对松浦镜鲤(Cyprinus carpio specularis)口灌烟酸诺氟沙星后,药物在实验鱼血浆中的药动学特征。3种给药剂量下,诺氟沙星在松浦镜鲤血浆中的血药浓度和时间关系均可用一级吸收二室开放模型进行描述,吸收半衰期(t1/2ka)分别为0.165、0.061、0.043 h,消除半衰期(t1/2β)分别为18.282、29.969、42.051 h,达峰时间(Tmax)分别为0.333、0.327、0.302 h,达峰浓度(Cmax)分别为4.780、6.247、12.689 mg/L,药时曲线下面积(AUC)分别为32.698、53.015、174.998 mg·h/L,表观分布容积(Vd)分别为1.044、4.347、4.561 L/kg。说明随着给药剂量的增加,诺氟沙星的吸收和消除速率均加快,给药剂量对药动学特征有显著影响。     

鲤鱼（Cyprinus carpio）生长激素基因的亚克隆及其序列分析

本文对ＰＣＲ扩增的６６８ｂｐ的ＤＮＡ片段进行了亚克隆,然后以Ｓａｎｇｅｒ双脱氧中止法为原理,利用美国ＡＢＩ公司３７０Ａ自动核酸序列分析仪,确定了６６８ｂｐ的核苷酸序列。序列分析表明鲤鱼生长激素基因的开放读框含有６３０ｂｐ,并推测其中包括２２个氨基酸的信号肽和１８８个氨基酸的成熟多肽。鲤鱼生长激素基因的酶切图谱和序列分析的结果都证明我们已获得了全长的鲤鱼生长激素基因。     

转基因红鲤体细胞的核移植

以F4代转hGH基因红鲤体细胞（肾脏和尾鳍）及培养18代的F4代转hGH基因红鲤尾鳍细胞为核供体,泥鳅或黄河鲤成熟卵为受体,进行了核移植,以探讨外源F4代转基因鱼体外源基因的分布与存在形式,稳定性和克隆转基因鱼的可能性。F4代红鲁肾脏细胞核与泥鳅卵配合的核移植胚胎有12．4％发育到囊胚,0．33％发育到神经胚;F4代尾鳍细胞核移入泥鳅卵后的重组胚发育到囊胚,神经胚、肌节期和肌肉效应期的胚胎分别为24．5％、0．3％、0．2％和0．1％;对照卵无发育。F4代红鲤尾鳍培养细胞与黄河鲤卵子配合的重组胚胎有50．53％发育到囊胚,5．69％发育到原肠胚,0．53％发育到神经胚,0．4％发育到肌节期。说明由于同种细胞核与卵细胞的相容性高于异种核卵的相容性,早期发育率高;而由于培养细胞的异倍化,后期的发育率降低。用PCR技术对供体鱼不同个体及同一体不同组织外源基因检测,结果100％个体为阳性鱼,而且不同组织的阳性率也是100％,说明外源基因均匀分布在不同组织中。无论F4代转基因鱼的肾脏细胞、尾鳍细胞还是培养的尾鳍细胞作核移植供体,核移植胚胎中hGH基因的检出率为100％。说明F4代转基因红鲤个体不同细胞都存在hGH基因,而且经长期培养不会丢失。表明F4代转基因红鲤中的外源hGH基因已基本稳定,体细胞核移植可以作为获得同质化转基因鱼的有效手段,但核移植效率还很低。另外还讨论了核质的相容性、细胞周期的协调、染色体的变异等因素对核移植的影响。     

建鲤(Cyprinus carpio var. Jian)微卫星DNA亲权鉴定

利用16个微卫星座位对建鲤10个全同胞家系647个子代进行亲权鉴定。Cervus3.0分析表明,16个微卫星位点的平均多态信息含量为0.7025,平均等位基因数为6.63,期望杂合度平均为0.7405。当双亲未知时,累积排除概率为0.999225,已知单亲时的累积排除概率为0.999996,置信度为95%。进一步模拟分析表明,要达到亲权鉴定的要求在双亲未知时通常需要8～12个微卫星位点,已知单亲时需要5～8个微卫星位点。在双亲均未知的情况下进行亲权鉴定,94.6%的后裔找到了其父母本,真实鉴定率低于模拟分析预测值,分析可能是与候选亲本间存在亲缘关系、无效等位基因的存在以及分型错误等因素有关。9个建鲤全同胞家系的鉴定,为今后的遗传图谱构建、QTL定位及分子标记辅助育种研究奠定了基础。     

Rheological Characteristics and Microstructure of Common Carp (Cyprinus carpio) Surimi and Kamaboko Gel

Common carp is cheap and prolific in Australian waters and is regarded as an aquatic environmental pest. In order to add value to this fish species, surimi and kamaboko was prepared from common carp and its rheological and microstructure characteristics were compared with those produced from Alaska pollock and threadfin bream. Temperature sweep tests were run under 100-Pa stress and 0.1-Hz frequency, obtained from linear viscoelastic ranges of all tested fish gels. Storage modulus (G′) thermographs of all samples consisted of two positive peaks and a plateau zone in between. The sol-gel transition was completed at about 53 and 61 °C for these Alaska pollock and threadfin bream gels, respectively, whereas it was recorded at about 69 °C for common carp gel. At these temperatures, G′ of Alaska pollock gel was recorded at 330 kPa, which was 71% and 88% greater than that from threadfin bream and common carp gels, respectively. Furthermore, Alaska pollock and threadfin bream gels had greater gel strength than the gel prepared from common carp surimi. Textural quality could be associated with cross-linking in the gel network. From scanning electron microscopy studies, the microstructure of Alaska pollock gel matrix had about 15,450 polygonal structures per square millimeter with an average area of about 9 μm². For threadfin bream and common carp gels, the polygonal structures were larger and 12% and 39% fewer, respectively, than those of Alaska pollock gel. However, these results cannot be only attributed to the species variation among tested fish as some other factors such as harvest location, physiological state, handling and processing method, etc. were not considered in this study.     

Carp (Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay

Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp.     

Toxic Effects of Cobalt Chloride on Hematological Factors of Common Carp (Cyprinus carpio)

In this study, we investigate the toxic effects of cobalt chloride on some hematological factors of the carp Cyprinus carpio, such as white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. At first, LC₅₀ of cobalt in C. carpio was measured during 96 h after exposure. Also, physicochemical parameters of water including pH, dissolved oxygen, viscosity, temperature, and conductivity were monitored, continuously. The results showed that LC₅₀ values of cobalt in C. carpio were 327 and 328 mg/L in two replicates, respectively. Then, the changes in some hematological factors in the five treatment groups placed under concentration of 100, 200, 300, 400, and 500 mg/L cobalt were compared with the control group. Based on hematological tests conducted in this research, exposure of carp to 500- and 300-mg/L concentrations of cobalt in 48 h showed significant difference (p?<?0.05) in white blood cell count. The concentration of 500 mg/L cobalt in 24 h showed a significant difference in the amount of hemoglobin, number of red blood cells, and hematocrit level as compared with the control group. The concentration of 100 mg/L cobalt in 48 h did not show a significant difference in comparison with the control group (p?>?0.05). Also, the concentration of 500 mg/L cobalt in 24 h showed a significant difference in the amount of mean corpuscular volume and mean corpuscular hemoglobin as compared with the control group and other treatments. Also, the percentage of mean corpuscular hemoglobin concentration in a concentration of 200 mg/L cobalt in 24 h showed a significant difference as compared with the control group and other treatments.     

NADPH-Diaphorase in the Olfactory System of the Carp Cyprinus carpio

Ultrastructural distribution of NADPH-diaphorase (NADPH-d) in olfactory epithelium and bulb of the carp Cyprinus carpio L. was studied using light and electron microscopy. The diaphorase staining was revealed in the supranuclear area of the sensory and indifferent epithelium, in the olfactory nerve, as well as in the outer layers of the olfactory bulb—in fibers and glomeruli. NADPH-d-positive neurons were found in the interglomerular neuropil. Electron microscopy showed that NADPH-d in the olfactory lining epithelium was related only to receptor cells and ciliary supporting cells and was present in submembranous structures. Besides, in both parts of the olfactory system the main, cytosolic part of the enzyme is bound to cytoskeleton and is also present in membranes of endoplasmic reticulum and in mitochondria. In general, the NADPH-d of the carp olfactory system is characterized by predominantly intracellular localization and widespread contacts of the enzyme with cytosol.     

Impact of introduced carp (Cyprinus carpio)in subtropical shallow ponds in Central Mexico

In Mexico, as in many other subtropical and tropical countries, there has been a recent trend towards stocking non-native carp (Cyprinus carpio) in lakes and ponds as a source of food in rural areas. However, the results of a study in a series of small(1–8 ha.), shallow (<2 m), semi-natural ponds in Acambay, a high altitude valley in the basin of the Lerma river in the volcanic belt in central west Mexico, illustrate that the stocking of carp over a threshold value may have a detrimental ecological impact at several trophic levels. Ponds with carp tended to be turbid with high levels of suspended solids, and with few rooted macrophytes and epibenthic invertebrates. In contrast, ponds without carp had clear water and abundant rooted macrophytes and associated invertebrates, particularly gastropod molluscs. The direct uprooting of macrophytes by benthic foraging carp appeared to be the most important mechanism in switching the ponds from a clear macrophyte-dominated to a turbid state. The subtropical study ponds thus appear to confirm the alternative stable-state hypothesis developed intemperate lakes, although the importance of benthic rather than pelagic interactions was emphasised. The implications of stocking carp for native fauna of high intrinsic conservation value and as a food supply for local people are outlined. This revised version was published online in July 2006 with corrections to the Cover Date.     

锦鲤中吉氏库特菌的分离与鉴定

对从患病锦鲤(Cyprinus carpio L.)肝组织中分离的1株纯培养菌(HC050630C-1)进行了形态特征、主要理化特性、对健康鲤鱼的致病作用、药物敏感性等方面的检验;同时测定了该株菌的16S rRNA基因序列,构建了系统发育树。结果表明,被检菌为吉氏库特菌(Kurthia gibsonll),对健康鲤鱼未显示明显的致病作用;所测菌株的16S rRNA基因序列长度1459bp,在GenBank中登录号为EF611423;该菌株16SrRNA基因序列与GenBank数据库中吉氏库特菌的16S rRNA基因序列同源性在99%;药敏试验结果显示,对供试的氨苄青霉素等27种药物呈现高度敏感或敏感,对头孢吡肟等6种呈低度敏感,对苯唑青霉素等4种耐药。     

Chronic Exposure to Tributyltin Induces Brain Functional Damage in Juvenile Common Carp (Cyprinus carpio)

The aim of the present study was to investigate the effect of Tributyltin (TBT) on brain function and neurotoxicity of freshwater teleost. The effects of long-term exposure to TBT on antioxidant related indices (MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; GPx, glutathione peroxidase), Na⁺-K⁺-ATPase and neurological parameters (AChE, acetylcholinesterase; MAO, monoamine oxidase; NO, nitric oxide) in the brain of common carp were evaluated. Fish were exposed to sublethal concentrations of TBT (75 ng/L, 0.75 μg/L and 7.5 μg/L) for 15, 30, and 60 days. Based on the results, a low level and short-term TBT-induced stress could not induce the notable responses of the fish brain, but long-term exposure (more than 15 days) to TBT could lead to obvious physiological-biochemical responses (based on the measured parameters). The results also strongly indicated that neurotoxicity of TBT to fish. Thus, the measured physiological responses in fish brain could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity.     

Carp (Cyprinus carpio) muscle fructose 1,6-bisphosphatase: purification and some properties.

1. Fructose 1,6-bisphosphatase from the white muscle tissue of the carp, Cyprinus carpio L. was purified. 2. The mol. wt of the enzyme was 145,000. Its subunit mol. wt was ca. 35,000. 3. The enzyme exhibited neutral pH optimum, activation by monovalent cations, and temperature-dependent allosteric AMP inhibition. 4. Carp muscle fructose 1,6-bisphosphatase was 10- to 30-fold more sensitive to AMP inhibition than the carp liver enzyme. 5. The carp muscle enzyme was less sensitive to AMP inhibition than the muscle enzyme from a homeothermic mammal. These results are interpreted as an example of temperature-adaptation of an enzyme regulatory property.